This invention relates to a novel class of phosphonic acid derivatives that are inhibitors of PTP-1B.
Protein tyrosine phosphatases are a large family of transmembrane or intracellular enzymes that dephosphorylate substrates involved in a variety of regulatory processes (Fischer et al., 1991, Science 253:401-406). Protein tyrosine phosphatase-1B (PTP-1B) is a xcx9c50 kd intracellular protein present in abundant amounts in various human tissues (Charbonneau et al., 1989, Proc. Natl. Acad. Sci. USA 86:5252-5256; Goldstein, 1993, Receptor 3:1-15).
Determining which proteins are substrates of PTP-1B has been of considerable interest. One substrate which has aroused especial interest is the insulin receptor. The binding of insulin to its receptor results in autophosphorylation of the receptor, most notably on tyrosines 1146, 1150, and 1151 in the kinase catalytic domain (White and Kahn, 1994, J. Biol. Chem. 269:1-4). This causes activation of the insulin receptor tyrosine kinase, which phosphorylates the various insulin receptor substrate (IRS) proteins that propagate the insulin signaling event further downstream to mediate insulin""s various biological effects.
Seely et al., 1996, Diabetes 45:1379-1385 (xe2x80x9cSeelyxe2x80x9d) studied the relationship of PTP-1B and the insulin receptor in vitro. Seely constructed a glutathione S-transferase (GST) fusion protein of PTP-1B that had a point mutation in the PTP-1B catalytic domain. Although catalytically inactive, this fusion protein was able to bind to the insulin receptor, as demonstrated by its ability to precipitate the insulin receptor from purified receptor preparations and from whole cell lysates derived from cells expressing the insulin receptor.
Ahmad et al., 1995, J. Biol. Chem. 270:20503-20508 used osmotic loading to introduce PTP-1B neutralizing antibodies into rat KRC-7 hepatoma cells. The presence of the antibody in the cells resulted in an increase of 42% and 38%, respectively, in insulin stimulated DNA synthesis and phosphatidyinositol 3xe2x80x2 kinase activity. Insulin receptor autophosphorylation and insulin receptor substrate-1 tyrosine phosphorylation were increased 2.2 and 2.0-fold, respectively, in the antibody-loaded cells. The antibody-loaded cells also showed a 57% increase in insulin stimulated insulin receptor kinase activity toward exogenous peptide substrates.
Recently, Kennedy et al., 1999, Science 283: 1544-1548 showed that protein tyrosine phosphatase PTP-1B is a negative regulator of the insulin signalling pathway, suggesting that inhibitors of this enzyme may be beneficial in the treatment of Type 2 diabetes. Mice lacking PTP-1B are resistant to both diabetes and obesity.
Thus, inhibitors of PTP-1B improve insulin-sensitivity. They have utility in controlling or treating Type 1 and Type 2 diabetes, in improving glucose tolerance, and in improving insulin sensitivity in patients in need thereof. The compounds may also be useful in treating or preventing cancer, neurodegenerative diseases and the like.
Compounds represented by Formula I, including pharmaceutically acceptable salts thereof, and prodrugs thereof, are PTP-1B inhibitors and are useful in the treatment of diabetes, obesity, and related conditions. 
In these compounds, R1 and R2 are selected from the group consisting of C1-10alkyl(Ra)0-7, C2-10alkenyl(Ra)0-7, Aryl(Ra)0-3 and Het(Ra)0-3;
wherein, each Ra independently represents a member selected from the group consisting of: Aryl, OH, halogen, C0-6alkyleneCO2H, C0-6alkyleneCO2C1-6alkyl, OC1-10alkyl, C1-6alkyl, C1-6haloalkyl, OC1-10alkyleneCO2H, S(O)yC1-6alkyl, S(O)yNR3xe2x80x2R4xe2x80x2, and Het, wherein y is 0, 1, or 2;
Ar represents Aryl or Het, wherein said Aryl or Het is substituted with 1-5 substituents Rb, wherein optionally 2 Rb groups can join together to form a 5-7 membered ring fused to Ar, where the fused portion of the 5-7 membered ring may be saturated or may include 1-2 double bonds and may include 1-4 heteroatoms selected from N, S, O, and C(xe2x95x90O) in the fused portion of the ring, said ring optionally being substituted with 1-3 groups independently selected from Ra;
Aryl is a 6-14 membered carbocyclic aromatic ring system comprising 1-3 phenyl rings, wherein said rings are fused together when there is more than one aromatic ring;
Het represents a 5-10 membered aromatic ring system comprising one ring or two fused rings, 1-4 heteroatoms, 0-4 of which are N atoms and 0-2 of which are or S(O)y wherein y is 0-2, and 0-2 carbonyl groups;
Each Rb is independently selected from the group consisting of: OH, CN, halogen, C0-6alkyleneOC1-6alkyl(Ra)0-7, C0-6alkyleneOAryl(Ra)0-3, Het, C0-6alkyleneS(O)yC1-6alkyl(Ra)0-7, with y equal to 0-2, C0-6alkyleneS(O)3H, C1-10alkyl(Ra)0-7, N3, C0-6alkyleneCO2H, C0-6alkyleneCO2C1-6alkyl(RA)0-7, C0-6alkyleneCO2C2-6 alkenyl(Ra)0-7, C0-6alkyleneC(O)C1-6alkyl(Ra)0-7, C(O)NR3xe2x80x2R4xe2x80x2, S(O)yNR3xe2x80x2R4xe2x80x2, NR3xe2x80x2R4xe2x80x2, PO(OR5)2, and CF2PO(OR5)2, wherein R3xe2x80x2 and R4xe2x80x2 are as defined above;
Each y is 0, 1 or 2;
Each R5 is H;
Y1, Y2, Z1 and Z2 each independently represents xe2x80x94(CR3R4)a-X-(CR3R4)bxe2x80x94 wherein a and b are each zero or an integer 1 or 2 such that the sum of a and b equals 0, 1, 2 or 3,
X represents a bond, O, S(O)y, NR3xe2x80x2, C(O), OC(O), C(O)O, C(O)NR3xe2x80x2, NR3xe2x80x2(O) or xe2x80x94CHxe2x95x90CHxe2x80x94, where y is as previously defined;
R3 and R4 are independently H, halogen, C1-10alkyl or C1-10haloalkyl;
R3xe2x80x2 is selected from the group consisting of: H, C1-6alkyl, C1-6haloalkyl, OH, C(O)C1-6 alkyl, C(O)Aryl, C(O)Het, C(O)C1-6 haloalkyl, Aryl and Het;
R4xe2x80x2 is selected from the group consisting of: H, C1-6alkyl, C1-6haloalkyl, Aryl and Het;
Each W1 is independently selected from the group consisting of: H, OH, CN, halogen, OC1-6alkyl(Ra)0-7, OAryl(Ra)0-3, S(O)yC1-6alkyl(Ra)0-7, with y equal to 0-2, S(O)3H, C1-6alkyl(Ra)0-7, N3, C0-6alkyleneCO2H, C0-6alkyleneCO2C1-6 alkyl (Ra)0-7, C0-6alkyleneCO2C2-6 alkenyl(Ra)0-7, C0-6alkyleneC(O)C1-6alkyl(Ra)0-7, C(O)NR3xe2x80x2R4xe2x95x90, S(O)yNR3xe2x80x2R4xe2x80x2, NR3xe2x80x2R4xe2x80x2, Aryl and Het, wherein R3xe2x80x2and R4xe2x80x2 are as defined above; or alternatively two W1 groups on adjacent atoms of the aromatic ring are joined together to form a fused phenyl ring, optionally substituted with 1-3 groups Rb.
Methods of treating, controlling, and preventing diabetes, obesity, and other related diseases and conditions using the compounds having Formula I are provided herein. Pharmaceutical compositions and combination therapies are also disclosed herein.
In one embodiment of this invention, in compounds having Formula I, W1 is independently selected from the group consisting of:
(a) hydrogen,
(b) halogen,
(c) OC1-6alkyl(Ra)0-7,
(d) SC1-6alkyl(Ra)0-7,
(e) C1-6alkyl(Ra)0-7,
(f) CO2H,
(g) CO2-C1-6alkyl(Ra)0-7,
(h) OH,
(l) N(R3xe2x80x2)(R4xe2x80x2) and
(m) C(O)C1-6alkyl(Ra)0-7.
In another embodiment, each W1 represents H or halogen.
In another embodiment, Ar represents phenyl, quinolinyl, indolyl or thienopyridinyl.
In a subset of compounds having Formula I, as described above, Ar represents phenyl, which is substituted with 1-2 substituents selected from Rb, and the phenyl ring to which Ar is connected is unsubstituted.
In a preferred subset of compounds of Formula I, Y1, Z1 and Z2 are each independently CH2 or a bond, and Y2 is selected from CH2, a bond, xe2x80x94CH2CH2SCH2xe2x80x94, xe2x80x94CH2CH2OH2xe2x80x94, xe2x80x94C(xe2x95x90O)OCH2xe2x80x94, xe2x80x94C(xe2x95x90O)OCH2CH2xe2x80x94, and xe2x80x94C(xe2x95x90O)Oxe2x80x94.
In another subset of compounds of Formula I, Rb is selected from the group consisting of: halogen, C0-6alkyleneOC1-6alkyl(Ra)0-2, xe2x80x94SC1-6alkyl(Ra)0-2, -Ophenyl, tetrazole, C1-10alkyl(Ra)0-2, C0-3alkyleneCO2H, C0-3alkyleneCO2C1-6alkyl(Ra)0-2, C(O)NR3xe2x80x2R4xe2x80x2, S(O)yNR3xe2x80x2R4xe2x80x2, PO(OR5)2, and CF2PO(OR5)2, wherein R3xe2x80x2 and R4xe2x80x2 are individually selected from H and C1-6alkyl, and Ra is selected from OH, xe2x80x94OC1-3alkyl, and phenyl.
In another group of compounds, R1 and R2 are selected from Aryl(Ra)0-3 and Het(Ra)0-3.
In many preferred compounds, R1 and R2 are selected from phenyl and 1H-1,2,3-Benzotriazolyl.
Finally specific compounds of Formula I are provided in Table 1, Table 2, and Examples 1-27. The compounds in Examples 1-27 are named below:
Methods of treating, preventing, or controlling diabetes and other diseases using the compounds of Formula I are disclosed herein. A method of treating, controlling or preventing diabetes and complications thereof in a mammalian patient in need of such treatment includes the administration to the patient an anti-diabetic effective amount of a compound of Formula I. A method of treating, controlling or preventing obesity in a mammalian patient in need of such treatment comprises the administration to the patient an anti-obesity effective amount of a compound in accordance with claim 1. Such methods also include the administration of a second compound, which may be an anti-diabetic compound, an anti-obesity compound, or an HMG-CoA reductase inhibitor, in an amount effective to treat, control or prevent diabetes or obesity, or to improve a poor lipid profile.
A method of treating, controlling or preventing atherosclerosis in a mammalian patient in need of such treatment comprises administering to the patient an effective amount of a compound of Formula I and an effective amount of an HMG-CoA reductase inhibitor.
More generally, compounds of Formula I may be used as the active compound in a method for treating, preventing, or controlling one or more diseases or conditions selected from Type 1 diabetes, Type 2 diabetes, inadequate glucose tolerance, insulin resistance, obesity, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, atherosclerosis, vascular restenosis, inflammatory bowel disease, pancreatitis, adipose cell tumors, adipose cell carcinoma, liposarcoma, dyslipidemia, cancer, and neurodegenerative disease. The method comprises the administration of an effective amount of the compound of Formula I. Combination treatments can also be used in which case, the method comprises the administration of a compound of Formula I and an effective amount of one or more pharmaceutically active compounds selected from the group consisting of an HMG-CoA reductase inhibitor, an anti-obesity agent, and an antidiabetic compound.
Pharmaceutical compositions also can be made using the compounds of Formula I. Compositions that are suitable for the treatment, prevention or control of one or more diseases or conditions selected from Type 1 diabetes, Type 2 diabetes, inadequate glucose tolerance, insulin resistance, obesity, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, atherosclerosis, vascular restenosis, inflammatory bowel disease, pancreatitis, adipose cell tumors, adipose cell carcinoma, liposarcoma, dyslipidemia, cancer, and neurodegenerative disease contain an effective amount of a compound of Formula I in combination with a pharmaceutically acceptable carrier.
Such pharmaceutical compositions may also include a second anti-diabetic agent or an anti-obesity agent. They may also include a cholesterol lowering agent. Pharmaceutical compositions may therefore include: (1) an effective amount of a compound of Formula I, (2) an effective amount of one or more pharmaceutically active compounds selected from the group consisting of an HMG-CoA reductase inhibitor, an anti-obesity agent, and an anti-diabetic agent, and (3) a pharmaceutically acceptable carrier.
Such pharmaceutical compositions that contain a second active compound or composition and that are suitable for the treatment, prevention or control of one or more diseases or conditions selected from the group consisting of Type 1 diabetes, Type 2 diabetes, inadequate glucose tolerance, insulin resistance, obesity, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, atherosclerosis, vascular restenosis, inflammatory bowel disease, pancreatitis, adipose cell tumors, adipose cell carcinoma, liposarcoma, dyslipidemia, cancer, and neurodegenerative disease, may be comprised of the following:
(1) an effective amount of a compound of Formula 1;
(2) an effective amount of one or more pharmaceutically active compounds listed below; and
(3) a pharmaceutically acceptable carrier; where the pharmaceutically active compounds are selected from the group consisting of:
(a) insulin sensitizers including (i) PPARxcex3 agonists such as the glitazones (e.g. troglitazone, pioglitazone, englitazone, MCC-555, rosiglitazone, and the like), and compounds disclosed in WO97/27857, 97/28115, 97/28137 and 97/27847; (ii) biguanides such as metformin and phenformin;
(b) insulin or insulin mimetics;
(c) sulfonylureas such as tolbutamide and glipizide, or related materials;
(d) xcex1-glucosidase inhibitors (such as acarbose);
(e) cholesterol lowering agents such as (i) HMG-CoA reductase inhibitors (lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, rivastatin and other statins), (ii) sequestrants (cholestyramine, colestipol and a dialkylaminoalkyl derivatives of a cross-linked dextran), (iii) nicotinyl alcohol, nicotinic acid or a salt thereof, (iv) PPARxcex1 agonists such as fenofibric acid derivatives (gemfibrozil, clofibrate, fenofibrate and bezafibrate), (v) inhibitors of cholesterol absorption, for example beta-sitosterol, and acyl CoA:cholesterol acyltransferase inhibitors, for example melinamide and (vi) probucol;
(f) PPARxcex1/xcex3 agonists;
(g) antiobesity compounds such as appetite suppressants, fenfluramine, dexfenfluramine, phentiramine, sulbitramine, orlistat, neuropeptide Y5 inhibitors (NP Y5 receptor antagonosts), leptin, which is a peptidic hormone, xcex23 adrenergic receptor agonists, and PPARxcex3 antagonists and partial agonists;
(h) ileal bile acid transporter inhibitors; and
(i) insulin receptor activators.
Abbreviations
The following abbreviations have the indicated meanings:
AA=arachidonic acid
Ac=acetyl
AIBN=2.2xe2x88x92-azobisisobutyronitrile
Bn=benzyl
BSA=bovine serum albumin
Bz=benzoyl
CHO=chinese hamster ovary
CMC=1-cyclohexyl-3-(2-morpholinoethyl) carbodiimidemetho-p-toluenesulfonate
DAST=diethylamino sulfur trifluoride
DBU=diazabicyclo[5.4.0]undec-7-ene
DMAP=4-(dimethylamino)pyridine
DMF=N,N-dimethylformamide
DMSO=dimethyl sulfoxide
Et3N=triethylamine
HATU=O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HBSS=Hanks balanced salt solution
HEPES=N1-[2-Hydroxyethyl]piperazine-N4-[2-ethanesulfonic acid]
HWB=human whole blood
KHMDS=potassium hexamethyldisilazane
LDA=lithium diisopropylamide
LPS=lipopolysaccharide
mCPBA=metachloro perbenzoic acid
MMPP=magnesium monoperoxyphthalate
Ms=methanesulfonyl=mesyl
MsO=methanesulfonate=mesylate
NBS=N-bromosuccinimide
NCS=N-chlorosuccinimiide
NIS=N-iodosuccinimide
NSAID=non-steroidal anti-inflammatory drug
Oxone(copyright)=potassium peroxymonosulfate
PCC=pyridinium chlorochromate
PDC=pyridinium dichromate
PPA=polyphosphoric acid
PTP=protein tyrosine phosphatase
r.t.=room temperature
rac.=racemic
Tf=trifluoromethanesulfonyl=triflyl
TFA=trifluoroacetic acid
TFAA=trifluoroacetic anhydride
TfO=trifluoromethanesulfonate=triflate
THF=tetrahydrofuran
TLC=thin layer chromatography
Ts=p-toluenesulfonyl=tosyl
TsO=p-toluenesulfonate=tosylate
Tz=1H (or 2H)-tetrazol-5-yl
Alkyl Group Abbreviations
Me=methyl
Et=ethyl
n-Pr=normal propyl
i-Pr=isopropyl (occasionally written xe2x80x9cprixe2x80x9d)
n-Bu=normal butyl
i-Bu=isobutyl
s-Bu=secondary butyl
t-Bu=tertiary butyl
c-Pr=cyclopropyl
c-Bu=cyclobutyl
c-Pen=cyclopentyl
c-Hex=cyclohexyl
Dose Abbreviations
bid=bis in die=twice daily
qid=quater in die=four times a day
tid=ter in die=three times a day
Alkyl means linear, branched and cyclic structures, and combinations thereof, containing the indicated number of carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4-propylnonyl, cyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl, 2-ethyl-1- bicyclo[4.4.0]decyl and the like.
Fluoroalkyl means alkyl groups of the indicated number of carbon atoms in which one or more hydrogens is replaced by fluorine. Examples are xe2x80x94CF3, xe2x80x94CH2CH2F, xe2x80x94CH2CF3, c-Pr-F5, c-Hex-F11 and the like. Haloalkyl has the analogous meaning for replacement of one or more hydrogen atoms with any halogen (Cl, Br, F, and/or I).
Alkenyl means linear, branched and cyclic structures, and combinations thereof containing a double bond with the indicated number of carbon atoms. Examples of alkenyl groups include allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 2-methyl-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl and the like. Alkadienyl means the diunsaturated counterpart to alkenyl.
Alkynyl means linear, branched and cyclic structures, and combinations thereof containing a triple bond with the indicated number of carbon atoms. Examples of alkynyl groups include propargyl, 2-butynyl, 3-butynyl, 2-pentynyl, cyclopropylethynyl, and the like.
Alkylene, alkenylene, alkynylene, fluoroalkylene, alkadienylene, and the like, where the suffix xe2x80x9cenexe2x80x9d has been added to the name of the monovalent radicals alkyl, alkenyl, alkynyl, fluoroalkyl, alkadienyl, and the like, describe divalent radicals that are the same as their monovalent counterparts, except that two hydrogen atoms rather than one are removed so that the radical will have two attachments.
Aryl means a 6-14 membered carbocyclic aromatic ring system comprising 1-3 phenyl rings. If two or more aromatic rings are present, then the rings are fused together, so that adjacent rings share a common side.
Heteroaryl (Het) as used herein represents a 5-10 membered aromatic ring system containing one ring or two fused rings, 1-4 heteroatoms, 0-4 of which are N atoms and 0-2 of which are 0 or S(O)y wherein y is as previously defined, and 0-2 carbonyl groups. Carbonyl groups, when present, are not counted as heteroatoms. Het includes, but is not limited to, furanyl, diazinyl, imidazolyl, isooxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyrazolyl, pyridyl, pyrrolyl, tetrazinyl, thiazolyl, thienyl, triazinyl, triazolyl, 1H-pyrrole-2,5-dionyl, 2-pyrone, 4-pyrone, pyrrolopyridine, furopyridine and thienopyridine.
Benzoheteroaryl, which is a subset of Het includes aromatic ring systems containing one or more heteroatoms which also have a fused 6-membered benzene ring, such as 2H-1-benzopyran-2-one, 4H-1-benzopyran-4-one, 2(3H)benzofuranone, 3(2H)benzofuranone, 2,3-dihydrobenzofuran, 2,3-dihydrobenzothiophene, indole, benzofuran, benzothiophene, benzimidazole, benzoxazole, benzothiazole, benzotriazole, benzothiadiazole, 1H-isoindole-1,3(2H)-dione, quinoline, and isoquinoline.
Another subset of heteroaryls includes 5-membered heteroaryls, such as the following: 
When a heteroaromatic ring is specified as optionally having one or more heteroatoms, this means that at least one heteroatom is present, selected from O, S and N, and up to 4 such heteroatoms may be present, depending upon the size of the ring specified.
When a moiety is specified as being optionally substituted, then the same moiety may also remain unsubstituted, unless otherwise stated.
Finally, when a list of possible choices is provided for a given moiety, and the moiety is used in more than one position in a chemical formula, the selection of a choice for the moiety in each position is independent of other selections, unless the definition says otherwise.
Metabolitesxe2x80x94Prodrugs
Metabolites of the compounds of this invention that are therapeutically active and that are described by formula I also are within the scope of the claimed invention, as are prodrugs, which are compounds that are converted to the claimed active compounds or salts of the claimed active compounds after they have been administered to a patient. A non-limiting example of a prodrug of the phosphonic acids of this invention would be a monoester or diester of one or more phosphonic acid groups, where the ester functionality preferably has a structure that makes it easily hydrolyzed or metabolized after administration to a patient. Examples of prodrugs include C1-6 alkyl esters of the phosphonic acids. Prodrugs that have structures that are more easily hydrolyzed or metabolized are generally more preferred. Examples are illustrated by the structures below, where Rxe2x80x2xe2x95x90H or a C1-6alkyl group, and Rxe2x80x3xe2x95x90C1-6 alkyl group or xe2x80x94OC1-6 alkyl group, and Q is the residue of the molecule that is attached to the xe2x80x94CF2PO3H2 or xe2x80x94PO3H2 group in formula I. The alkyl groups and alkoxy groups may optionally be substituted with one or more substituents independently selected from 1-5 halogen atoms, a phenyl group, or a mixture of these. The phenyl group, if present, may optionally be substituted with 1-3 substituents independently selected from halogen, xe2x80x94CH3, xe2x80x94CF3, xe2x80x94OCH3 and xe2x80x94OCF3. In these compounds, and as defined in general throughout this application, the alkyl groups and the alkyl portions of Oalkyl groups may be linear or branched and may optionally be cycloalkyl or may include a cycloalkyl group in their structure. For examples of prodrug structures related to those shown below, see D. N. Srinivasta et al., Bioorganic Chemistry 12, 118-129 (1984). 
Other ester functionalities that may be used in the monoester or diester phosphonate prodrugs include phenyl esters and benzyl esters, where the phenyl ester groups have the structure -Ophenyl, and the benzyl ester groups have the structure xe2x80x94OCHRxe2x80x2phenyl, in which R is H or C1-6alkyl, and C1-6alkyl is substituted as described above. In either case, phenyl is substituted as described above.
The prodrugs of this invention may therefore be defined as compounds having the formula I, in which at least one group R5 is selected from the group consisting of C1-6alkyl, phenyl, xe2x80x94CHRxe2x80x2phenyl, and xe2x80x94CHRxe2x80x2OC(xe2x95x90O)Rxe2x80x9d, and the remaining groups R5 are selected from H, C1-6alkyl, phenyl, xe2x80x94CHRxe2x80x2phenyl and xe2x80x94CHRxe2x80x2OC(xe2x95x90O)Rxe2x80x3, wherein each group Rxe2x80x2 is H or C1-6alkyl and each group Rxe2x80x3 is xe2x80x94C1-6alkyl or xe2x80x94OC1-6alkyl, where C1-6alkyl and the alkyl portion of xe2x80x94OC1-6alkyl may optionally be substituted with one or more substituents independently selected from 1-5 halogen atoms, a phenyl group, or a mixture of these. The phenyl group in xe2x80x94CHRxe2x80x2phenyl, the phenyl group that is an optional substituent on C1-6alkyl and xe2x80x94OC1-6alkyl, and the phenyl ester group that is obtained when R5 is phenyl may optionally be substituted with 1-3 groups independently selected from halogen, xe2x80x94CH3, xe2x80x94CF3, xe2x80x94OCH3 and xe2x80x94OCF3. By this definition, at least one of the phosphonic acid groups is a monoester or diester, and each of the remaining phosphonic acid groups, if any, may be a free acid or a monoester or diester.
In preferred compounds, the groups R5 that are not H may all be the same because of the difficulty of synthesizing different R5 groups on the same phosphonates. In many cases, the prodrug will be a mixture of compounds having different levels of esterification on the phosphonic acid groups because of the difficulty of synthesizing and separating a discrete pure compound.
Optical Isomersxe2x80x94Diastereomersxe2x80x94Geometric Isomers
Some of the compounds described herein contain one or more asymmetric centers, and these asymmetric centers may give rise to diastereomers and enantiomers, which may be in the form of enantiomeric or diastereomeric mixtures or of individual optical isomers. The present invention includes all such diastereomers and enantiomers, including racemic mixtures and resolved, enantiomerically pure forms, and pharmaceutically acceptable salts thereof. Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, include both E and Z geometric isomers.
Salts
The pharmaceutical compositions of the present invention comprise a compound of the current invention as an active ingredient or a pharmaceutically acceptable salt, thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,Nxe2x80x2-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable acids, including inorganic and organic acids. Such acids include acetic, adipic, aspartic, 1,5-naphthalenedisulfonic, benzenesulfonic, benzoic, camphorsulfonic, citric, 1,2-ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, fumaric, glucoheptonic, gluconic, glutamic, hydriodic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, 2-naphthalenesulfonic, nitric, oxalic, pamoic, pantothenic, phosphoric, pivalic, propionic, salicylic, stearic, succinic, sulfuric, tartaric, p-toluenesulfonic acid, undecanoic, 10-undecenoic, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, methanesulfonic, phosphoric, sulfuric and tartaric acids.
It will be understood that in the discussion of methods of treatment or of specific compounds which follows, references to the compounds of Formula I and other formulae are meant to include the pharmaceutically acceptable salts.
Utilities
Inhibitors of PTP-1B improve insulin-sensitivity and thus have utility in preventing or treating Type 1 and Type 2 diabetes, improving glucose tolerance and insulin-sensitivity when there is insulin-resistance, and in treating or preventing obesity, all in mammals that are in need of such treatments or that might benefit from such treatments. The compounds also exhibit a beneficial reduction in triglycerides and lipids. Compounds in the present class of phosphonic acids are advantageous over known phosphonic acids previously investigated as candidate PTP-1B inhibitors. The compounds of this invention show greater selectivity for PTP-1B over T-Cell Protein Tyrosine Phosphatase (TCPTP) when compared with known phosphonates. This advantage minimizes possible toxicity due to the inhibition of TCPTP activity. These compounds are also active in intact cell-based assays.
The PTP-1B inhibitors may also be useful in the treatment, prevention or control of a number of conditions that accompany type 2 diabetes, including hyperlipidemia, hypertriglyceridemia, hypercholesterolemia (including beneficially raising low HDL levels), atherosclerosis, vascular restenosis, pancreatitis, adipose cell tumors, adipose cell carcinomas such as liposarcoma, dyslipidemia, inflammatory bowel disease, inflammation in general, and other disorders where insulin resistance is a component. Finally, the compounds may be used to treat or prevent cancer, such as prostate cancer, neurodegenerative diseases and the like.
Pharmaceutical Compositions
For the treatment of any of these PTP-1B-mediated diseases the active compound may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage units containing conventional pharmaceutically acceptable carriers. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular and intrasternal injection and infusion techniques. In addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, etc., the compounds of the invention are useful for the treatment of humans.
The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or miscible solvents such as propylene glycol, PEGs and ethanol, or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethycellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxy-ethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Examples of vehicles and solvents include water, Ringer""s solution and isotonic sodium chloride. Cosolvents such as ethanol, propylene glycol or polyethylene glycols may also be used. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds may also be administered in the form of suppositories. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but molten at the body temperature and will therefore release the drug. Such materials include cocoa butter and polyethylene glycols.
For topical use, creams, ointments, gels, solutions or suspensions containing the compound are employed. (For purposes of this application, topical application includes mouth washes and gargles.) Topical formulations may include cosolvents, emulsifiers, penetration enhancers, preservatives, emollients and the like.
The pharmaceutical composition may also be further comprised of a second anti-diabetic or anti-obesity effective compound.
Dose Ranges
Dosage levels on the order of from about 0.01 mg to about 100 mg/kg of body weight per day are useful in the treatment of the above-indicated conditions, or alternatively about 0.5 mg to about 7 g per patient per day. For example, the diseases and conditions described herein may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day.
The active ingredient is typically combined with the carrier to produce a dosage form suitable for the particular patient being treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from about 0.5 mg to about 5 g of the active agent, compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Representative dosage forms will generally contain between from about 1 mg to about 500 mg of an active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.
It is understood that the specific dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Combinations with Other Drugs
In further aspects, the invention encompasses pharmaceutical compositions for treating PTP-1B mediated diseases as defined above comprising an effective amount of the active compound and one or more other pharmaceutically active compounds, such as anti-diabetic compounds (for example, insulin, sulfonyl ureas, PPAR-alpha and/or -gamma ligiands, including ligands that have both PPAR-alpha and -gamma activity), anti-obesity compounds, and compounds that improve the lipid profile of the patient.
Thus, the methods of treatment or prevention described herein may further be comprised of administering to said patient a second anti-diabetic compound in an amount effective to treat, control, or prevent diabetes, alone or in combination with the PTP-1B inhibitors of this invention.
Similarly, the methods of treatment or prevention described herein may further be comprised of administering to said patient an anti-obesity compound in an amount effective to treat, control or prevent obesity, alone or in combination with the PTP-1B inhibitors of this invention.
Similarly, the methods of treatment of diabetes may comprise the administration of a cholesterol biosynthesis inhibitor, particularly an HMG-CoA reductase inhibitor, such as lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rivastatin, in an amount effective to improve the lipid profile. In combination with a PTP-1B inhibitor, this may be beneficial in treating or preventing atherosclerosis and other conditions that often are associated with Type 2 diabetes.
Examples of other pharmaceutically active compounds that may be combined with a compound of Formula I and administered in combination with the PTP-1B inhibitors include, but are not limited to, the following compounds or compositions or groups of compounds or compositions that are used as anti-diabetes compounds (a, b, c, d, f, and i below), anti-obesity compounds (g below), and/or compounds or compositions for lipid profile control (e and h below):
(a) insulin sensitizers including (i) PPARxcex3 agonists such as the glitazones (e.g. troglitazone, pioglitazone, englitazone, MCC-555, rosiglitazone, and the like), and compounds disclosed in WO97/27857, 97/28115, 97/28137 and 97/27847; (ii) biguanides such as metformin and phenformin;
(b) insulin or insulin mimetics;
(c) sulfonylureas such as tolbutamide and glipizide, or related materials;
(d) xcex1-glucosidase inhibitors (such as acarbose);
(e) cholesterol lowering agents such as (i) HMG-CoA reductase inhibitors (lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, rivastatin and other statins), (ii) sequestrants (cholestyramine, colestipol and a dialkylaminoalkyl derivatives of a cross-linked dextran), (iii) nicotinyl alcohol, nicotinic acid or a salt thereof, (iv) PPARxcex1 agonists such as fenofibric acid derivatives (gemfibrozil, clofibrate, fenofibrate and benzafibrate), (v) inhibitors of cholesterol absorption for example beta-sitosterol and (acyl CoA:cholesterol acyltransferase) inhibitors for example melinamide and (vi) probucol;
(f) PPARxcex1/xcex3 agonists;
(g) antiobesity compounds such as appetite suppressants, fenfluramine, dexfenfluramine, phentiramine, sulbitramine, orlistat, neuropeptide Y5 inhibitors (NP Y5 receptor antagonosts), leptin, which is a peptidic hormone, xcex23 adrenergic receptor agonists, and PPARxcex3 antagonists and partial agonists;
(h) ileal bile acid transporter inhibitors; and
(i) insulin receptor activators, such as those disclosed in copending, commonly assigned U.S. application Ser. Nos. 09/095,244 and 09/280,602.
Where a second pharmaceutical is used in addition to an active compound taught herein, the two pharmaceuticals may be administered together in a single composition, separately at approximately the same time, or on separate dosing schedules. The important feature is that their dosing schedules comprise a treatment plan in which the dosing schedules overlap in time and thus are being followed concurrently.
Methods of Synthesis
The compounds of the present invention can be prepared according to the following methods.
Method A
Di-tert-butyl phosphite can be deprotonated with a base such as LiN(TMS)2 and reacted with a tolualdehyde to provide alcohol 2, which may be oxidized with MnO2 or under Swern""s conditions to give phosphonoketone 3. Bromination of 3 with NBS followed by fluorination with DAST affords bomide 5, which can be used for the alkylation of various 1-benzyl-1H-benzotriazole to give compound 7. Alkylation of 7 under the same condition with an appropriately substituted biphenyl benzyl bromide 8 gives compound 9 which hydrolyses under acid conditions to give product 10. 
Method B
Compound 7 can be alkylated with a bromosubstituted benzyl halide to give compound 11 which can then undergo a palladium catalyzed coupling reaction with a substituted phenylboronic acid to give compound 9. 
Method C
Compound 7 can be alkylated with a substituted biphenyl aldehyde 12 to give compound 13, acid hydrolysis of compound 13 gives product 14. 
Method D
Alkylation of benzotriazole with methyl 2-bromophenylacetate 15 gives the ester 16. Hydrolysis of ester 16 followed by alkylation of the corresponding acid with the benzyl alcohol gives the benzyl ester 17. Alkylation of benzyl ester 17 with the phosphonobenzyl bromide 5 gives compound 18. Hydrogenation of the benzyl ester 18 in the presence of palladium gives the corresponding acid 19 which can be alkylated with a bromomethylbiphenyl 8 to give the ester 20. Acid hydrolysis of ester 20 gives the product 21. 
Many methods are available for the synthesis of halomethybiphenyl 8. Some of the methods are described below.
Method E
Reaction of diiodobenzene with diethylphosphine in the presence of palladium catalyst gives the iodophosphonate 23. Coupling of the iodide 23 with hydroxymethylboronic acid 24 in the presence of palladium gives the corresponding hydroxymethylbiphenyl 25 which can be converted to the corresponding halide using POCl3/DMF. 
Method F
Palladium catalyzed coupling of 4-methylphenylboronic acid 27 with substituted tert-butyl-3-iodobenzoate 28 gives the corresponding biphenyl product which can be brominated by NBS to give the bromomethylbiphenyl derivative 29. 
Method G
Monoalkylation of methyl dihydroxybenzoate with an alkyl halide gives a mixture of monoalkoxy hydroxybenzoate 31. The hydroxybenzoate 31 can be converted to the corresponding triflate 32 which can undergo palladium catalyzed coupling reaction with 4-methylphenylboronic acid to give the biphenyl derivative 33. The methyl ester can then be exchanged to the corresponding t-butyl ester 34. Bromination of 34 gives the bromomethylbiphenyl derivative 35. 
Method H
Reaction of 2-methyl-6,8-dibromoquinoline with diethylphosphite in the presence of palladium catalyst gives a mixture of the corresponding 6- and 8-phosphonate 37 and 38 respectively. Coupling of the 8-phosphonate 38 with 4-hydroxymethylphenylboronic acid in the presence of palladium catalyst gives the corresponding biaryl derivative 39. The benzyl alcohol 39 can be converted to the corresponding benzyl bromide 40 using POBr3/DMF.
Compound 38 can be oxidized to the aldehyde 41 using SeO2. The aldehyde 41 can react with various Grignard reagents and the resulting alcohol can be protected as the methyl ether 42 or the methylmethyl ether 45.Compound 42 and 45 can be converted to the biaryl benzyl bromide 44 and 46 respectively using the same procedure described for transforming compound 38 to 40. 
Method I
Bromination of methyl thieno[3,2-b]pyridine-5-carboxylate gives the corresponding 3-bromo derivative 48. Palladium catalyzed coupling of compound 48 with 4-hydroxymethylphenylboronic acid gives the biaryl benzyl alcohol 49 which can be converted to the corresponding bromide 50 using POBr3/DMF. 
Method J
Palladium catalyzed coupling of 2-methyl-6,8-dibromo quinoline gives predominantly the 6-(hydroxymethylphenyl)quinoline derivative 52. The benzyl alcohol can be protected as the THP ether 53, which can be alkylated sequentially with various alkyl halides to give the mono- and dialkylated product 54 and 55 respectively. Palladium catalyzed coupling of diethylphosphite with 55 gave 56. Deprotection of the THP ether with HCl/EtOH, followed by bromination of the alcohol with POBr3 gives the bromide 57. 
Method K
4xe2x80x2-Methoxy-4-methylbiphenyl 58 is prepared from the Suzuki reaction of 4-methylbenzeneboronic acid and 4-bromoanisole. The methyl ether is then cleaved with a Lewis acid such as BBr3. The hydroxy intermediate is converted to the phosphate intermediate 60 followed Pumanand""s condition (Tet. Lett., 1989, 30, 1687). Base promoted rearrangement with a base such as LDA provides the hydroxy phosphonate intermediate 61. Alkylation with an alkyl halide in the presence of a base such as NaOH in a solvent such as DMF gives an alkoxy phosphonate intermediate 62. Bromination with NBS provides the bromomethyl intermediate 63 for subsequent alkylation reaction. 
Method L
The compound 66 can be obtained by the reaction of the thiolate generated in situ from the thiosilane 64, with an appropriate electrophile. The resulting intermediate 65 is then converted to the free phosphonic acid by treatment with a reagent such as TMSiBr. 
Methods M, N and O describe methods of making prodrugs from phosphonic acids and phosphonic acid salts. In Methods M, N and O, Q is the residue of the molecule that is attached to the xe2x80x94CF2PO3H2 group.
Method M
The disodium phosphonate 67 can be alkylated with a chloroalkyl ester (Synth. Com. 25(18) 2739 (1995)) or carbonate (Antiviral Chemistry and Chemotherapy 8, 557 (1997)) to give both the mono and diprotected phosphonates (68 and 69) which can be separated by flash chromatography on silica gel. 
The phosphonic acid 70 can be treated with Cs2CO3 and a chloroalkyl ester or carbonate in CH3CN to give a mixture of mono and diprotected phosphonates which can be separated by flash chromatography on silica gel. 
Method O
The phosphonic acid 70 can be treated with silver trifluoroacetate to give the disilver salt 71 which can be treated with an iodoalkyl ester (Eur. J. Phar. Sci. 4, 49 (1996)) or carbonate to give a mixture of the mono and diprotected phosphonates which are separable by flash chromatography. 
Activity in the compounds of this application is demonstrated using the following assays for PTP-1B-inhibiting activity.
Phosphatase Assay Protocol
Materials:
EDTA-ethylenediaminetetraacetic acid (Sigma)
DMH-N,Nxe2x80x2-dimethyl-N,Nxe2x80x2-bis(mercaptoacetyl)-hydrazine (synthesis published in J. Org. Chem. 56, pp. 2332-2337,(1991) by R. Singh and G. M. Whitesides and can be substituted with DTT -dithiothreitol Bistris -2,2-bis(hydroxymethyl)2,2xe2x80x2,2xe2x80x3-nitrilotriethanol-(Sigma) Triton X-100-octylphenolpoly(ethylene-glycolether) 10 (Pierce).
Antibody: Anti-glutathione S-transferase rabbit (H and L) fraction (Molecular Probes)
Enzyme: Human recombinant PTP-1B, containing amino acids 1-320, fused to GST enzyme (glutathione S-transferase) or to FLAG peptide purified by affinity chromatography (Huyer et al, 1997, J. Biol. Chem., 272, 843-852). Wild type contains active site cysteine(215), whereas mutant contains active site serine(215).
Tritiated peptide: Bz-NEJJ-CONH2, Mwt. 808, empirical formula, C32H32T2O12P2F4 
IC50 Binding Assay Protocol:
Compounds (ligands) which potentially inhibit the binding of a radioactive ligand to the specific phosphatase are screened in a 96-well plate format as follows:
To each well is added the following solutions @ 25xc2x0 C. in the following chronological order:
1. 110 xcexcl of assay buffer.
2. 10 xcexcl. of 50 nM tritiated BzN-EJJ-CONH2 in assay buffer (1xc3x97) @ 25xc2x0 C.
3. 10 xcexcl. of testing compound in DMSO at 10 different concentrations in serial dilution (final DMSO, about 5% v/v) in duplicate @ 25xc2x0 C.
4. 10 xcexcl. of 3.75 xcexcg/ml purified human recombinant GST-PTP-1B in enzyme dilution buffer.
5. The plate is shaken for 2 minutes.
6. 10 xcexcl. of 0.3 xcexcg/ml anti-glutathione S-transferase (anti-GST) rabbit IgG (Molecular Probes) diluted in antibody dilution buffer @ 25xc2x0 C.
7. The plate is shaken for 2 minutes.
8. 50 xcexcl. of protein A-PVT SPA beads (Amersham) @ 25xc2x0 C.
9. The plate is shaken for 5 minutes. The binding signal is quantified on a Microbeta 96-well plate counter.
10. The non-specific signal is defined as the enzyme-ligand binding in the absence of anti-GST antibody.
11. 100% binding activity is defined as the enzyme-ligand binding in the presence of anti-GST antibody, but in the absence of the testing ligands with the non-specific binding subtracted.
12. Percentage of inhibition is calculated accordingly.
13. IC50 value is approximated from the non-linear regression fit with the 4-parameter/multiple sites equation (described in: xe2x80x9cRobust Statisticsxe2x80x9d, New York, Wiley, by P. J. Huber (1981) and reported in nM units.
14. Test ligands (compounds) with larger than 90% inhibition at 10 xcexcM are defined as actives.
Enzyme Assay PTP-1B
Assay buffer 50 mM Bis-Tris (pH=6.3)
2 mM EDTA
5 mM N,Nxe2x80x2-dimethyl-N,Nxe2x80x2-bis(mercaptoacetyl)hydrazine (DMH)
Substrate 10 mM fluorescein diphosphate (FDP) store at xe2x88x92200C
Enzyme dilution buffer 50 mM Bis-Tris (pH=6.3)
2 mM EDTA
5 mM DMH
20%(v/v) glycerol
0.01% Triton X-100
The assay was carried out at room temperature in 96 well plates. The reaction mixture in 170 xcexcl contained 50 mM Bis-Tris (pH=6.3), 2 mM EDTA, 5 mM N,Nxe2x80x2-dimethyl-N,Nxe2x80x2bis(mercaptoacetyl)hydrazine (DMH) and 10 xcexcM fluorescein diphosphare (FDP). 10 xcexcl of 10 concentrations (serial dilution) of the test compound (inhibitor) dissolved in DMSO or DMSO alone for control was added to each well and the plate was mixed for 2 min. The reaction was initiated by adding 20 xcexcl of diluted PTP-1B (50 nM in 50 mM Bis/Tris (pH=6.3), 2 mM EDTA, 5 mM DMH, 20% glycerol and 0.01% Triton X-100. The phosphatase activity was followed by monitoring the appearance of the fluorescent product fluorescein monophosphate (FMP) continuously for 15-30 min, using the Cytofluor II plate reader (PerSeptive Biosystems Inc.) with excitation of 440 nm (slit width 20 nm) and emission at 530 nm (slit width 25 nm). All the assays were done at least in duplicate. The initial rate of FMP formation is plotted against the concentration of inhibitor and the data was fitted to 4-parameter equation and the inflection point of the fit is the IC50.
Pharmacokinetics in Rats
Per Os Pharmacokinetics in Rats
Procedure:
The animals are housed, fed and cared for according to the Guidelines of the Canadian Council on Animal Care.
Male Sprague Dawley rats (325-375 g) are fasted overnight prior to each PO blood level study.
The rats are placed in the restrainer one at a time and the box firmly secured. The zero blood sample is obtained by nicking a small (1 mm or less) piece off the tip of the tail. The tail is then stroked with a firm but gentle motion from the top to the bottom to milk out the blood. Approximately 1 mL of blood is collected into a heparinized vacutainer tube.
Compounds are prepared as required, in a standard dosing volume of 10 mL/kg, and administered orally by passing a 16 gauge, 3xe2x80x3 gavaging needle into the stomach.
Subsequent bleeds are taken in the same manner as the zero bleed except that there is no need to nick the tail again. The tail is cleaned with a piece of gauze and milked/stroked as described above into the appropriately labelled tubes.
Immediately after sampling, blood is centrifuged, separated, put into clearly marked vials and stored in a freezer until analysed.
Typical time points for determination of rat blood levels after PO dosing are:
0, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h
After the 4 hr time point bleed, food is provided to the rats ad libitum. Water is provided at all times during the study.
Vehicles:
The following vehicles may be used in PO rat blood level determinations:
PEG 200/300/400: restricted to 2 mL/kg
Methocel 0.5%-1.0%: 10 mL/kg
Tween 80: 10 mL/kg
Compounds for PO blood levels can be in suspension form. For better dissolution, the solution can be placed in a sonicator for approximately 5 minutes.
For analysis, aliquots are diluted with an equal volume of acetonitrile and centrifuged to remove protein precipitate. The supernatant is injected directly onto a C-18 HPLC column with UV detection. Quantitation is done relative to a clean blood sample spiked with a known quantity of drug. Bioavailability (F) is assessed by comparing area under the curve (AUC) i.v. versus p.o.   F  =            AUCpo      AUCiv        xc3x97          DOSEiv      DOSEpo        xc3x97    100    ⁢          xe2x80x83        ⁢    %  
Clearance rates are calculated from the following relation:   CL  =            DOSEiv      ⁡              (                  mg          /          kg                )              AUCiv  
The units of CL are mL/hxc2x7kg (milliliters per hour kilogram)
Intravenous Pharmacokinetics in Rats
Procedure:
The animals are housed, fed and cared for according to the Guidelines of the Canadian Council on Animal Care.
Male Sprague Dawley (325-375 g) rats are placed in plastic shoe box cages with a suspended floor, cage top, water bottle and food.
The compound is prepared as required, in a standard dosing volume of 1 mL/kg.
Rats are bled for the zero blood sample and dosed under CO2 sedation. The rats, one at a time, are placed in a primed CO2 chamber and taken out as soon as they have lost their righting reflex. The rat is then placed on a restraining board, a nose cone with CO2 delivery is placed over the muzzle and the rat restrained to the board with elastics. With the use of forceps and scissors, the jugular vein is exposed and the zero sample taken, followed by a measured dose of compound which is injected into the jugular vein. Light digital pressure is applied to the injection site, and the nose cone is removed. The time is noted. This constitutes the zero time point.
The 5 min bleed is taken by nicking a piece (1-2 mm) off the tip of the tail. The tail is then stroked with a firm but gentle motion from the top of the tail to the bottom to milk the blood out of the tail. Approximately 1 mL of blood is collected into a heparinized collection vial. Subsequent bleeds are taken in the same fashion, except that there is no need to nick the tail again. The tail is cleaned with a piece of gauze and bled, as described above, into the appropriate labelled tubes.
Typical time points for determination of rat blood levels after I.V. dosing are either:
0, 5 min, 15 min, 30 min, 1 h, 2 h, 6 h
or
0, 5 min, 30 min, 1 h, 2 h, 4 h, 6 h.
Vehicles:
The following vehicles may be used in IV rat blood level determinations:
Dextrose: 1 mL/kg
2-Hydroxypropyl-b-cyclodextrin 1 mL/kg
DMSO (dimethylsulfoxide): Restricted to a dose volume of 0.1 mL per animal
PEG 200: Not more than 60% mixed with 40% sterile waterxe2x88x921 mL/kg
With Dextrose, either sodium bicarbonate or sodium carbonate can be added if the solution is cloudy.
For analysis, aliquots are diluted with an equal volume of acetonitrile and centrifuged to remove protein precipitate. The supernatant is injected directly onto a C-18 HPLC column with UV detection. Quantitation is done relative to a clean blood sample spiked with a known quantity of drug. Bioavailability (F) is assessed by comparing area under the curve (AUC) i.v. versus p.o.   F  =            AUCpo      AUCiv        xc3x97          DOSEiv      DOSEpo        xc3x97    100    ⁢          xe2x80x83        ⁢    %  
Clearance rates are calculated from the following relation:   CL  =            DOSEiv      ⁡              (                  mg          /          kg                )              AUCiv  
The units of CL are mL/hxc2x7kg (milliliters per hour kilogram).
This assay is the subject of copending, commonly assigned U.S. Provisional Application No. 60/123,243, filed Mar. 8, 1999, which patent application is incorporated herein by reference, and was recently published in Cromlish, Wanda A., Paul Payette and Brian P. Kennedy (1999) Biochem Pharmocol 58: 1539-1546.
Construction of Recombinant Baculovirus Transfer Vectors and Insect Cells
Briefly, using the Bac-to-Bac Baculovirus Expression System (Gibco-BRL, Mississauga, Ontario, Canada) PTP 1B cDNA (obtained from Dr. R. L. Erikson, Harvard University, USA), is cloned into the pFASTBAC donor plasmid engineered to include a FLAG sequence at the 5xe2x80x2 end of the cDNA (PTP1B-FL). The recombinant plasmid is transformed into competent DH10BAC E. Coli cells. Following transposition and antibiotic selection, the recombinant bacmid DNA is isolated from selected E. Coli colonies and used to transfect sf9 insect cells (Invitrogen, San Diego, Calif., U.S.A.). The sf9 cells are cultured in spinner flasks at 28xc2x0 C. in Graces supplemented medium (Gibco-BRL, Mississauga, Ontario, Canada) with 10% heat-inactivated fetal bovine serum (Gibco-BRL) following the protocol of Summers and Smith (A manual for Methods for Baculovirus Vectors and Insect Culture Procedures(Bulletin No. 1555). Texas A and M University, Texas Agricultural Experiment Station, College Station, Tex., 1987).
Intact Cell Assay
Infected sf9 cells expressing PTP1B-FL and mock infected cells, are harvested at 29 hpi (hours post infection) by gentle centrifugation (Beckman GS-6R) at 460 rpm, (48 g) for 5 min. Cells are washed once in assay buffer (Hanks"" solution buffered with 15 mM Hepes, pH 7.4, obtained from Sigma, St. Louis, Mo., U.S.A.) and recentrifuged at 300 rpm (21 g) for 10 min. The cells are then gently resuspended in assay buffer and examined using a hemacytometer for cell density and viability by trypan blue exclusion. Assays are performed using a Tomtec Quadra 96 pipeting robot, programmed to mix the cells gently after each addition. In 200 xcexcL of assay buffer, 2xc3x97105 PTP expressing cells or mock infected cells are dispensed into each well of 96-well polypropylene plates and pre-incubated either with a test compound or DMSO vehicle (3 xcexcL), for 15 min at 37xc2x0 C. The pre-incubated cells are challenged with a final concentration of 10 mM pNPP (p-nitrophenyl phosphate, obtained from Sigma-Aldrich Canada Ltd., Oakville, Ontario) for 15 min, centrifuged at 4xc2x0 C. and the amount of substrate hydrolysis is determined spectrophotometerically at OD405.
Oral Glucose Tolerance Test
Oral glucose tolerance tests are done on conscious Zucker obese fa/fa rats or obese ob/ob mice (age 12 weeks or older). The animals are fasted for 16-18 hours before use for experiments. A test compound or a vehicle is given either intraperitoneally or orally 60 minutes before oral administration of a glucose solution at a dose of 2 g/kg body weight. Blood glucose levels are measured using a Medisense glucometer from tail bled samples taken at different time points before and after administration of glucose. A time curve of the blood glucose levels is generated and the area-under-the-curve (AUC) for 120 minutes is calculated (the time of glucose administration being time zero). Percent inhibition is determined using the AUC in the vehicle-control group as zero percent inhibition.
In separate studies, C57BL/6J mice are fed a high fat (35%) and high carbohydrate (36%) diet obtained from Bioserv (Frenchtown, N.J.) for 3 to 4 weeks, at which time the mice gained 50-100% of the baseline body weight. Oral glucose tolerance tests are done in the same manner as described above.